Viability, ultrastructure and cytokinin metabolism of free and immobilized tobacco chloroplasts

Cytokinins play a decisive role in regulation of plastid development and differentiation, but their metabolism in plastids is not known. Metabolic studies using intact chloroplasts are prevented by their instability once they are isolated from leaf cells. Chloroplasts of Nicotiana tabacum L. cv. Petit Havana SR1 were therefore immobilized into low-viscosity alginate. Their intactness was assessed by a glyceraldehyde-3-phosphate dehydrogenase assay which indicated that free chloroplasts totally disintegrated within 7 h, while more than 50% of immobilized chloroplasts remained intact after 24 h. The immobilization had no marked impact on ultrastructure and postponed final destruction. The metabolite profile was similar in free and immobilized chloroplasts after 4 h incubation with tritiated zeatin. Nevertheless, the yield of conversion products decreased twice in immobilized chloroplasts, which was probably the outcome of mass transfer limitations and/or the sorption to polysaccharide matrix.     

Egg size and offspring performance in the collared flycatcher (<Emphasis Type="Italic">Ficedula albicollis</Emphasis>): a within-clutch approach

Adaptive within-clutch allocation of resources by laying females is an important focus of evolutionary studies. However, the critical assumption of these studies, namely that within-clutch egg-size deviations affect offspring performance, has been properly tested only rarely. In this study, we investigated effects of within-clutch deviations in egg size on nestling survival, weight, fledgling condition, structural size and offspring recruitment to the breeding population in the collared flycatcher (Ficedula albicollis). Besides egg-size effects, we also followed effects of hatching asynchrony, laying sequence, offspring sex and paternity. There was no influence of egg size on nestling survival, tarsus length, condition or recruitment. Initially significant effect on nestling mass disappeared as nestlings approached fledging. Thus, there seems to be limited potential for a laying female to exploit within-clutch egg-size variation adaptively in the collared flycatcher, which agrees with the majority of earlier studies on other bird species. Instead, we suggest that within-clutch egg-size variation originates from the effects of proximate constraints on laying females. If true, adaptive explanations for within-clutch patterns in egg size should be invoked with caution.     

High content of endogenous cytokinins stimulates activity of enzymes and proteins involved in stress response in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

Transgenic Pssu-ipt tobacco with elevated content of endogenous cytokinins grown under in vitro conditions exhibited elevated activities of antioxidant enzymes (i.e. catalase, ascorbate peroxidase, guaiacol and syringaldazine peroxidase, glutathione reductase) and some of enzymes involved in anaplerotic pathways such as glucose-6-phosphate dehydrogenase, glycolate oxidase, NADP-malic enzyme, NADP-isocitrate dehydrogenase, and glutamate dehydrogenase compared to control non-transgenic SR1 tobacco. Higher activities of peroxidases, NADP-malic enzyme, and glutamate dehydrogenase were maintained in transgenic grafts after several weeks of the growth under ex vitro conditions, while transgenic rooted plants showed only the increase in activity of glycolate oxidase compared to control non-transformed tobacco. Total activities of superoxide dismutase were lower in both types of Pssu-ipt tobacco contrary to controls under both growth conditions. The presence of PR-1 protein and proteins with elevated activities of chitinase was proved in the extracellular fluid in both transgenic types under both in vitro and ex vitro conditions.     

Exogenous administration of gangliosides inhibits Fc epsilon RI-mediated mast cell degranulation by decreasing the activity of phospholipase C gamma

Gangliosides released from tumor cells, as well as administered exogenously, suppress the immune responses by largely unknown mechanisms. We show here that a pretreatment of rat basophilic leukemia cells with isolated brain gangliosides inhibited the release of preformed secretory mediators from cells activated via FcepsilonRI but not Thy-1 glycoprotein. Exogenously administered gangliosides also affected the cell-substrate adhesion and the levels of polymeric filamentous actin in Ag-activated cells. Although the production of phosphoinositides was also decreased, enzymatic activity of phosphatidylinositol 3-kinase was not inhibited. Gangliosides had no or only marginal effect on the association of aggregated FcepsilonRI with glycosphingolipid-enriched membranes and on tyrosine phosphorylation of FcepsilonRI and the linker for activation of T cells. Though pretreatment with gangliosides did not inhibit the association of linker for activation of T cells with phospholipase C (PLC)gamma1 and PLCgamma2, tyrosine phosphorylation of these enzymes, as well as their enzymatic activities and association with detergent-insoluble signaling assemblies were reduced. This resulted in a decreased production of inositol 1,4,5-trisphosphate and an inhibition of Ca(2+) mobilization. The combined data support the concept that exogenously administered gangliosides interfere with those properties of glycosphingolipid-enriched membranes that are important for the formation of plasma membrane-associated signaling assemblies containing PLCgamma but not for initial tyrosine phosphorylation of FcepsilonRI subunits.     

Towards proteome database of Francisella tularensis

The accessibility of the partial genome sequence of Francisella tularensis strain Schu 4 was the starting point for a comprehensive proteome analysis of the intracellular pathogen F. tularensis. The main goal of this study is identification of protein candidates of value for the development of diagnostics, therapeutics and vaccines. In this review, the current status of 2-DE F. tularensis database building, approaches used for identification of biologically important subsets of F. tularensis proteins, and functional and topological assignments of identified proteins using various prediction programs and database homology searches are presented.     

Methionine proximity assay,a novel method for exploring peptide ligand-receptor interaction

Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p,p'-nitrobenzoylphenylalanine (NO2Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT1 and AT2, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.     

Juncus uruguensis—a member of the section Juncotypus (Juncaceae, Juncus subg. Agathryon)

Anatomy and morphology of Juncus uruguensis were studied in order to determine the sectional position of the species. Leaf anatomy, a decisive criterion for sectional classification in the subg. Agathryon , shows that the species should be placed in the sect. Juncotypus , not in the sect. Steirochloa.     

How does a wetland plant respond to increasing temperature along a latitudinal gradient?

Global warming affects plant fitness through changes in functional traits and thereby ecosystem function. Wetlands are declining worldwide, and hence, ecosystem functions linked to wetlands are threatened. We use Caltha palustris “a common wetland plant” to study whether warming affects growth and reproduction differently depending on origin of source population, potentially affecting phenotypic response to local climate. We conducted a 2‐year in situ temperature manipulation experiment using clone pairs of C. palustris in four regions, along a 1300‐km latitudinal gradient of Sweden. Open‐top chambers were used to passively increase temperature, paired with controls. Growth and reproductive traits were measured from 320 plants (four regions × five sites × two treatments × eight plants) over two consecutive seasons to assess the effect of warming over time. We found that warming increased plant height, leaf area, number of leaves, and roots. High‐latitude populations responded more strongly to warming than low‐latitude populations, especially by increasing leaf area. Warming increased number of flowers in general, but only in the second year, while number of fruits increased in low‐latitude populations the first year. Prolonged warming leads to an increase in both number of leaves and flowers over time. While reproduction shows varying and regional responses to warming, impacts on plant growth, especially in high‐latitude populations, have more profound effects. Such effects could lead to changes in plant community composition with increased abundance of fast‐growing plants with larger leaves and more clones, affecting plant competition and ecological functions such as decomposition and nutrient retention. Effects of warming were highly context dependent; thus, we encourage further use of warming experiments to predict changes in growth, reproduction, and community composition across wetland types and climate gradients targeting different plant forms.